Tight junctions (TJ) consist of a network of sealing strands that form selective ion permeable barriers between epithelial or endothelial cells. In the inner ear, TJs play an essential role in maintaining the separation between endolymph and perilymph. TJ strands are primarily composed of linear oligomers of members of the claudin (CLDN) protein family. We have recently demonstrated that CLDN strands are inherently flexible and identified a novel cis interaction (Cis-1) required for strand formation and structural flexibility. Using the CLDN15 strand model we show that residue R79 which is unique to Cis-1 is essential for normal strand formation. R81H missense mutation in CLDN14 has been associated with DFNB29 human deafness. CLDN14 R81 is located in the Cis-1 region and corresponds to CLDN15 R79. We studied the role of R81 in the formation and stability of CLDN14. The sequences of human and mouse CLDN14 were aligned and homology modeled on CLDN15. TJ formation and strand structure were examined by transfection of COS7 or HEK293T cells with fluorophore-tagged claudins. Confocal microscopy was used to determine the frequency of TJ formation between transfected cell pairs and expression of claudins in the plasma membrane of mutant cells. Freeze-fracture transmission electron microscopy was used to determine strand morphology. CLDN15 R79 mutants showed a reduced frequency of TJ formation between transfected cells compared to wild type CLDN15. Freeze-fracture of the CLDN15 R79A strands displayed normal morphology, suggesting that the residue is not essential for proper protein folding. Similarly, CLDN14 mutations R81H, R81W, and R81E all failed to form TJs. The plasma membrane expression of both mouse CLDN15 R79 mutants and CLDN14 R81 mutants was found equivalent or greater to wild-type, showing that plasma membrane expression levels of the mutants were not limiting TJ formation, and agreeing with the reported recessive nature of the R81H mutation. Transfected cell pairs co-expressing wild type and mutant CLDN14 R81H formed TJs, but the mutant was excluded from the TJ. These results show that R81 is important for CLDN14 strand formation and mutations can prevent Cis-1 interactions between Cldn protomers. In the inner ear, the R81H mutation likely causes a loss of the apical CLDN14 TJ strands and likely disrupting the endolymph/perilymph barrier. The proper connection between inner ear cochlear hair cell stereocilia and the overlaying tectorial membrane (TM) is critical for normal hearing. Outer hair cells (OHCs) physically interact with the lower side of the TM forming W-shaped imprints. The imprints correspond to the sites of OHC stereocilia-tectorial membrane junctions (STJs) required for synchronization and amplification of hair bundle deflection during sound-induced oscillations of the Organ of Corti. Mutant mouse models for several human deafness genes have revealed that proper stereocilia elongation and organization is critical for normal hearing. We used scanning electron microscopy to examine the alterations in STJ in several mouse models for hearing loss including mice with short stereocilia bundles, with planar cell polarity (PCP) disruption, and with stereocilia degeneration during aging. Adult mutants with very short stereocilia (e.g. Myo15a-/-, Eps8-/-) showed an overall well-preserved TM but no STJs. Whrn-/- knockout mice showed significantly shorter stereocilia along the apical-basal axis of the sensory epithelium and presented many STJ imprints in the apical turn, few in the middle, and sporadic in the basal region of the cochlea. Mice with PCP defects (e.g. Myo6-/-, NmIIc-/-, Pcdh15-Cd2-/-) or with disorganized bundle morphology (e.g. Sans-/-, Tmhs-/-) exhibit abnormal STJs in correspondence with their stereocilia morphology. In older mice, the TM surprisingly contains all the normal imprints from OHC stereocilia long after the disappearance of OHCs, revealing limited plasticity or turnover of the TM surface. Our data suggest that the coupling between stereocilia and tectorial membrane requires proper stereocilia elongation and precise bundle formation. We conclude that the lack of STJ in short stereocilia mutants is a key component of the pathophysiology of their associated mutations. The fact that the adult TM has limited plasticity is relevant to gene therapy and research on age-related hearing loss. The microvilli-rich surface of most epithelial cells is covered by a highly hydrated fibrous meshwork of glycoproteins generically called glycocalyx. In the vestibular sensory epithelium, the glycocalyx is involved in integrating the otoconial membrane to the microvilli of the supporting cells. It has also been shown that disruption of the glycocalyx affects the normal structure and function of the sensory stereocilia. Very little information is available on glycocalyx molecular architecture, mainly due to difficulties in preserving its delicate structure and in visualizing glycoproteins with conventional electron microscopy techniques. Here, we used enterocytes of the murine small intestine as a highly accessible and robust model system to study the detailed architecture of the glycocalyx, and how it is integrated to the surface of epithelial microvilli. We combined freeze-etch and electron tomography to describe in unprecedented details the organization of the microvilli-glycocalyx complex. We observed that the glycocalyx layer on the surface of enterocytes is made of very regular, 1.00.08 m-long, columnar filaments that emerge from the plasma membrane at the distal ends of the microvilli. Six to eight filaments emerge from each microvillus which in turn are highly cross-linked and hexagonally-packed with a center to center spacing of 16314 nm. The closely spaced columnar glycocalyx filaments make multiple anastomosing side-to-side interactions along their length over and across the surface of the enterocytes. The anastomosing produce filaments ranging from 3-12 nm in diameter and the mesh size of the network does not exceed 40 nm. Surprisingly, the glycocalyx filament termini come together in groups of three and four to form an ordered cover net that displayed liquid hexagonal packing with a center to center spacing of 328 nm. The microvilli, the columnar glycocalyx filaments and the terminal cover net form a stratified but highly integrated transcellular organization that maintains its regular structure across the enterocyte boundaries. Fourier analysis showed that despite the regular organization all three layers exhibit liquid packing properties. Our results provide a new structural framework for understanding how the microvilli-glycocalyx complex is organized and maintained. We will use these novel findings to reexamine and investigate further how the glycocalyx complex is involved in the development, maintenance, and function of the inner ear sensory epithelia.